The p53 protein is known as a "guardian of the genome" because of its crucial role in coordinating cellular responses to genotoxic stress. It is well accepted that Mdm2, a RING-finger E3 ubiquitin ligase, induces p53 ubiquitination and plays a major part in the scope of p53 regulation. Originally, the ubiquitin-proteasome pathway was thought to have a one way direction from substrate ubiquitination to degradation by the 26S proteasome. However, the discovery and emergence of deubiquitination enzymes (DUBs) changed the global view of the enzymatic process and quickly showed the incredible dynamics of this pathway. Our early finding that the Herpesvirus-Associated Ubiquitin-Specific Protease (HAUSP) interacts and stabilizes p53 by deubiquitination, was one of the first indications thatDUBs exhibited a specific role in the p53 pathway. Surprisingly, the simple linear model was obscured however with the subsequent findings that HAUSP deubiquitinates Mdm2 and is essential for controlling the Mdm2 stability in vivo. In addition to ubiquitinating p53, Mdm2 elicits high levels of self-ubiquitination which makes Mdm2 itself very liable in cells. Our studies demonstrate that HAUSP expression can rescue Mdm2 from self-ubiquitination. Moreover, SiRNA-mediated inactivation of endogenous HAUSP leads to unmanageable self-ubiquitination and destabilization of Mdm2, which indirectly results in p53 activation. These findings were further supported by the study of somatic HAUSP-knock out human cells (HCT116-HAUSP-/-) in Bert Vogelstein's lab and more recently confirmed in mouse HAUSP (-/-) embryos by our lab. Thus, our studies suggest a dynamic role of HAUSP-mediated deubiquitination in regulating the p53 pathway;however, it also raises more interesting questions regarding the precise function of HAUSP in vivo. For example, 1) what is the physiological role of HAUSP in p53 activation and tumorigenesis and 2) how are the HAUSP/Mdm2 and HAUSP/p53 interactions dynamically regulated? To understand the dynamic nature of this pathway, we will first try to elucidate the regulation of the HAUSP/Mdm2 interaction as well as the HAUSP/p53 interaction in vivo by posttranslational modifications (Aim1). To completely understand the biochemical role of HAUSP in p53 regulation, we plan to identify novel associated factor/ or regulators of the HAUSP complexes (Aim2). Finally, in Aim 3, we will develop a conditional HAUSP-null mouse to access the physiological role of HAUSP in the p53/Mdm2 pathway.